The purpose and scope of this project is subdivided into two specific aims that are detailed below: Specific Aim 1: Development of Site-Directed Photosensitized Labeling/crosslinking as a Tool to Study Membrane Protein Interactions We have developed a methodology that involves reaction of photo-activable probes with membrane proteins and lipids following activation of these probes in situ by energy transfer from a variety of donor chromophores. In the current studies we have used the membrane bilayer specific probe iodonaphthylazide (INA). We have used this method to establish which proteins of the viral envelope penetrate the target cell membrane in the course of infection and thus identify proteins and membrane compartments that participate in viral fusion. We monitored the insertion and redistribution of viral envelope proteins of orthomyxo, rhabdo, vaccinia and lentiviruses into the target cell membrane in the course of fusion. These studies shed light on portions of fusogenic proteins that insert into the viral and target membranes before, during and after fusion. We also established kinetic parameters of the intermediate stages in the fusion reaction and potentially resolve the route of viral entry (plasma membrane versus endosomal). We are also pursuing a photo affinity cross linking strategy to identify proteins involved in the mechanisms of HIV/SIV transmission through the viral synapse between dendritic cells and CD4+ lymphocytesSpecific Aim 2: Targeting the Hydrophobic Domain of Pathogens for Inactivation and Vaccine DevelopmentOur laboratory has developed a novel technology to inactivate a number of diverse enveloped pathogens. Our method uses photoactivatable hydrophobic compounds that, upon exposure to UV light, form nitrene radicals which react covalently with membrane proteins and lipids. This covalent modification has the ability to impair the function of multiple membrane proteins. We have focused on the impairment of membrane fusion mediated by viral envelope glycoproteins leading to inhibition of viral infection. We have been able to show the wide applicability of this inactivation technique to HIV, Influenza, Ebola, Marburg and VEE viruses, using compounds such as Iodonaphthyl azide (INA). By exclusively targeting the lipidic domain, exposed epitopes are preserved making the inactivated pathogens excellent vaccine candidates. Mouse studies have shown that immunization of mice with influenza virus inactivated with INA conferred heterosubtypic protection against the disease. We are extending the application of this immunization strategy to provide protection against pandemic viruses like avian influenza through our collaboration with the University of Georgia. We are investigating further improvements of our technology through the development of novel hydrophobic crosslinking agents. Results using 1,5-diazidonaphthalene (DAN) show that crosslinking reinforces the structure of the inactivated pathogens and confers some detergent resistance, potentially allowing for the purging of any residual infectious agents while preserving the integrity of the inactivated ones. This selective membrane stabilization followed by detergent treatment provides a method for orthogonal inactivation, which is key in producing safe, effective vaccines from whole inactivated pathogens. Viruses like particles (VLPs) are another approach to vaccine production, and would benefit from stabilization techniques to prolong their shelf-life. This technology is widely applicable to any enveloped pathogen in a very time efficient manner, which is critical in the case of emerging threats.